ABSTRACT Primary open-angle glaucoma is a leading cause of blindness worldwide. A primary risk factor for development and progression of glaucoma is elevation of intraocular pressure, caused by increased resistance in the aqueous humor outflow pathway. Most of the resistance is believed to be generated in the juxtacanalicular connective tissue (JCT) and modulated by the inner wall endothelium of Schlemm?s canal and its pores. The mechanisms that regulate aqueous outflow resistance remain unclear in normal and glaucomatous eyes, and only a couple newly-approved medications target this site of resistance. To traverse through the outflow pathway, aqueous humor passes through Schlemm?s canal endothelial cells in transient, pressure-driven cellular outpouchings, termed ?giant vacuoles,? (GV). Pores, small openings in GVs, allow aqueous humor to enter into Schlemm?s canal from the endothelium. In glaucoma, previous studies have shown a reduction in the number of GV and pores. The overall goal of this project is to understand the role of giant vacuoles and pores of Schlemm?s canal endothelial cells in regulating aqueous outflow. To do this, innovative technology will be used to observe and explore possibile factors (cytoskeletal dynamics and cellular connectivity) that influence the endothelial cells? ability to form giant vacuoles in new perspectives. First, a novel microfluidic device will be used in conjunction with live-cell imaging to capture real-time cytoskeletal dynamics of primary Schlemm?s canal endothelial cells in 3D cell culture. Secondly, serial block-face scanning electron microscopy will be used to analyze thousands of serial images along the inner wall of Schlemm?s canal of ex-vivo perfused human donor eyes. With 3D reconstruction of cellular geometries, the shapes of endothelial cells and their connections to underlying JCT cells and matrix will be analyzed and accurately quantified for the first time. Cells will be analyzed in different flow-types (high-, low-, and non-flow areas) based on fluorescent tracer distributions on global images to investigate how cellular connections affect the amount of flow going to certain regions around the circumference of the eye. The specific aims of this proposal are: 1) To observe cytoskeletal dynamics during giant vacuole formation in real time and determine monolayer permeability and hydraulic conductivity in response to a rho- kinase inhibitor or dexamethasone in primary Schlemm?s canal endothelial cells using a novel microfluidic device; and 2) To determine how cellular connections between Schlemm?s canal endothelial cells and underlying juxtacanalicular tissue cells/matrix modulate giant vacuole and pore formation and their contribution to regulation of segmental outflow using serial block-face scanning electron microscopy. The results of these studies will advance our understanding of the role of giant vacuoles and pores of Schlemm?s canal endothelial cells in regulating aqueous humor outflow. This new knowledge will help to develop new treatments for glaucoma by targeting the inner wall endothelium of Schlemm?s canal in the future.